Apoptosis-Modulating Protein Therapy for Proliferative Disorders and Nanoparticles Containing the Same

ABSTRACT

Protein containing nanoparticles and methods of use thereof for the treatment of proliferative disorders are disclosed.

This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application, 60/958,830 filed Jul. 9, 2007, the entire content of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the fields of drug delivery and proliferative disorders. More specifically, the invention provides p53 protein containing nanoparticles and methods of use thereof for the treatment of diseases associated with aberrant p53 functions, including without limitation, restenosis, tumor growth, for modulating drug effects which are dependent on p53 functional activity (e.g., drug resistance in cancer therapy), and altered artherogenesis.

BACKGROUND OF THE INVENTION

Several references and patent documents are cited throughout this application to better define the state of the art to which the invention pertains. Each of these citations is incorporated by reference herein as though set forth in full.

Gene delivery using non-viral systems such as liposomes and cationic lipid- or polymer-DNA complexes is usually transient and requires repeated delivery of the expression vector for the maintenance of a therapeutic level of the expressed protein in the target tissue (Li, S., Huang, L., (2000) Gene Ther. 7:31-34; Brown, M. D., et al, (2001) Int. J. Pharm. 229:1-21). The frequency of dosing of the expression vector, depending on the particular disease condition, depends on the efficiency of gene expression and the stability of the expressed protein in the tissue (Bonadio, I. et al., (1999), Nat. Med. 5:753-759). Repeated delivery of the vector may cause toxicity, including an inflammatory response and the therapy may not be effective (Maheshwari, A. et al. (2000), Mol. Ther. 2:121-130; Maheshwari, A. et al, (2002), Gene Ther. 9:1075-1084). To avoid these problems, various sustained release gene delivery systems such as polymeric implants and gels are being investigated (Bonadio, I. et al., (1999), Nat. Med. 5:753-759; Maheshwari, A.; et al. (2000), Mol. Ther. 2:121-130; Maheshwari, A. et al, (2002), Gene Ther. 9:1075-1084; Lim, Y. et al., (2000) Pharm. Res. 17: 811-816; Luo, D. at al. (1999) Pharm. Res. 16:1300-1308).

Recently, it has been demonstrated that nanoparticles (NPs) rapidly escape (within 10 min) from the endolysosomal compartment to the cytoplasmic compartment following their intracellular uptake via an endocytic process (Panyam. J. et al., (2002) FASEB J. 16:1217-1226). The escape of nanoparticles was attributed to the reversal of their surface charge from anionic to cationic in the acidic pH of the endolysosomal compartment, causing nanoparticles to interact with the endolysosomal membrane and then escape into the cytoplasmic compartment (Panyam. J. et al., (2002) FASEB J. 16:1217-1226). The rapid escape of nano-particles from the endolysosomal compartment could protect nanoparticles as well as the encapsulated therapeutic agent from the degradative environment of the endolysosomes (Prabha, S. et al., (2004) Pharm. Res. 21:354-363).

p53 is a well-studied protein, and its regulation is understood to play a significant role in cancer. The loss of p53 function is a very important event in cancer development. Data suggest that the absence of a functional p53 in tumors favors cancer development (Honda et al., (1998) Exp. Hematol. 26(3):188-97; Wiman, (1998) Med. Oncol. 15(4):222-8). p53 mutations are the most common genetic alterations observed in human cancers including lymphomas and leukemias (Shounan et al., (1997) Leukemia 11(10):1641-9; Chene, (2001) Curr. Med. Chem. Anticancer Agents. 1(2):151-61). It has been also suggested the loss of p53 function also affects the efficacy of anti-cancer drugs. Although several mechanisms are proposed, decreased p53 expression has been shown to result in increased extracellular matrix synthesis (Harisi et al., (2007) Cancer Biol. Ther. 6(8):1240-1246) or P-gp expression (Cavalcanti et al., (2004) Cytometry B Clin. Cytom. 61(1):1-8), thus reducing the drug uptake and hence the efficacy (e.g., doxorubicin).

In addition to cancer, p53 mutations play important role in other proliferative disorders. For example, Prolapsus uteri in pelvic support disorders are common in elderly women. It has been suggested that alterations in collagen synthesis and collagen types are related to this connective tissue disorder. The studies have shown that higher proliferative activity in prolapsus fibroblasts may result from the decreased expression of p53 protein and may lead to a decrease in the synthesis and deposition of extracellular matrix components (Yamamoto et al., (2000) Mech. Ageing Dev. 115(3):175-87). Another proliferative disorder, moyamoya, is a progressive cerebrovascular occlusive disease. It has been suggested that moyamoya disease may result, at least in part, from an abnormal regulation of extracellular matrix metabolism that leads to increased steady state levels of elastin mRNA and elastin accumulation in the initial thickening (Yamamoto et al., (1997) Stroke 28(9):1733-8).

‘Reactive species’ (RS) of various types are formed in vivo and many are powerful oxidizing agents, capable of damaging DNA and other biomolecules. Increased formation of RS can promote the development of malignancy, and the ‘normal’ rates of RS generation may account for the increased risk of cancer development in the aged. Hence additional actions of RS must be important, possibly their effects on p53, cell proliferation, invasiveness and metastasis (Halliwell, (2007) Biochem. J. 401(1):1-11).

Genetically manipulated mice with increased, but otherwise normally regulated, levels of Arf and p53 present strong cancer resistance and have decreased levels of ageing-associated damage. These observations extend the protective role of Arf/p53 to ageing, revealing a previously unknown anti-ageing mechanism and providing a rationale for the co-evolution of cancer resistance and longevity (Matheu et al., (2007) Nature. 448(7151):375-9).

Recent strategies have also turned to the p53 family member, p73, which like p53 is a potent inducer of death, but in contrast is rarely lost or mutated in tumors (Bell and Ryan (2007) Cell Cycle 6(16):1995-2000). p63 and p73, members of the p53 family, have been shown to be functionally distinct from p53. Based on gene sequence homologies, a p53 (TP53) gene family become apparent with the addition of the most recently identified p63 (TP73L; formerly TP63) and p73 (TP73) genes to the already known p53 (Kommagani et al., (2007) J. Biol. Chem. 282(41):29847-54). In addition to p73, p21 and p27 are other cell cycle proteins related to p53-mediated cell cycle arrest.

Delivery of wild type p53 encoding nucleic acid using a nanoparticle formulation has been successfully demonstrated, however, this system has certain drawbacks. For example, it is difficult to ensure that enough p53 nucleic acid enters the cell to be subsequently encoded into sufficient levels of functional p53 protein to ameliorate the symptoms of proliferative disease. Additionally, it is unclear whether diseased cells are capable of transcribing and producing protein in an efficient manner. It is an objective the present invention to provide an improvement to existing methods for delivery of p53, or other proteins involved in cellular senescence, to cells.

SUMMARY OF THE INVENTION

In accordance with the present invention, a method for inhibiting restenosis of a blood vessel (e.g., an artery or vein) comprising administering an effective amount of a protein containing nanoparticle via a blood vessel to a subject in need of treatment is provided. In a particular embodiment, the protein is selected from the group consisting of p21, p27, p53, p63, p73 or a functional fragment thereof. In another embodiment, the nanoparticle comprises a biodegradable polymer comprising a poly(lactide-co-glycolide), poly(lactic acid), poly(alkylene glycol), polybutylcyanoacrylate, poly(methylmethacrylate-co-methacrylic acid), poly-allylamine, polyanhydride, polyhydroxybutyric acid, or a polyorthoester or a combination thereof. In still another embodiment, the nanoparticle comprises a targeting moiety. In a different embodiment of the invention, the nanoparticle comprises a plasticizer.

In another aspect of the invention, a p53 protein nanoparticle formulation for sustained release of an effective amount of p53 protein said formulation comprising p53 protein, at least one biodegradable polymer, and an inert plasticizer are provided. In another aspect, the formulation further comprises an antioxidant, an anti-infective, an antiseptic, a steroid, a therapeutic peptide, an analgesic, an anti-inflammatory agent, an anticancer agent, a narcotic, an anesthetic, an antiangiogenic agent, a polysaccharide, a vaccine, an antigen, or a nucleic acid. In yet another aspect, the nanoparticle formulations also include a biodegradable polymer comprising a poly(lactide-co-glycolide), poly(lactic acid), poly(alkylene glycol), polybutylcyanoacrylate, poly(methylmethacrylate-co-methacrylic acid), poly-allylamine, polyanhydride, polyhydroxybutyric acid, or a polyorthoester. In a further aspect, the nanoparticle formulation comprises a targeting moiety.

The methods of the invention also include managing VSMC inflammation in a patient following angioplasty comprising administering to said patient a therapeutic agent in an effective amount to manage VSMC inflammation. In another embodiment, the therapeutic agent is a protein containing nanoparticle formulation. In yet another embodiment, the protein containing nanoparticle formulation contains a protein or protein fragment set forth in Table I or Table II.

In another aspect of the invention, a protein containing nanoparticle formulation wherein said protein is selected from the group consisting of SEQ ID NO: 1-28 in a pharmaceutically acceptable carrier is provided.

In yet another embodiment, a method of inhibiting inflammation in a patient following angioplasty is provided comprising administering to said patient a protein containing nanoparticle formulation comprising a protein or protein fragment selected from the group consisting of SEQ ID NO: 1-28.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram depicting the localization of nanoparticles in the arterial wall.

FIG. 2 shows the different domains of p53 and the p53 fragments described in Table I.

FIG. 3 is an SDS PAGE gel showing release of p53 from protein loaded NPs incubated in PBS in a double diffusion chamber. The receiver side of the buffer was withdrawn at different time intervals and analyzed. From left to right, Lane 1-200 ng of protein prior to entrapment in NPs; Lanes 2-5—p53 protein samples collected from the release study at day 1, day 3, day 7 and day 9 respectively.

FIG. 4, (A-D) are micrographs and graphs showing inhibition of restenosis with p53 protein therapy in a rat carotid artery model. FIG. 4A: Artery treated with control NPs; FIG. 4B: Artery treated with p53 protein-loaded modified NPs; FIG. 4C: Graph showing intima/media ratio between control and p53 protein treated cells; FIG. 4D: Graph showing lumen area in control vs. p53 protein treated cells.

DETAILED DESCRIPTION OF THE INVENTION

Although gene and drug therapy approaches have been extensively investigated for the inhibition of restenosis, there are no efforts to investigate protein therapy for this purpose. This could be due to a multitude of factors including (1) poor stability of proteins in the biological environment, (2) non-availability of a suitable carrier system that could deliver the protein effectively to a specific intracellular target, and (3) inability to maintain a therapeutic protein level in the target cells/tissue for the duration required to inhibit restenosis.

Nanoparticle formulations have been investigated that can release the encapsulated protein in active form over a period of time. The main therapeutic strategy to prevent post-angiopiasty restenosis has been to inhibit hyperplasia that is primarily caused by the migration and proliferation of vascular smooth muscle cells (VSMCs) and achieve vascular repair as indicated by re-endothelialization of the injured artery. Hyperplasia leads to re-obstruction of the injured artery in 30 to 50% patients undergoing balloon angioplasty. Over 1.5 million such procedures are performed annually worldwide, and in the US alone, 14 million people suffer from coronary artery disease, of which approximately 1 million undergo angioplasty annually. It is estimated that clinically significant restenosis continues to occur in >14% of elderly patients within the first year of undergoing coronary intervention procedure. This adds on average, $2747 per patient to the annual cost of follow-up care after angioplasty. Therefore, there are significant efforts to reduce the incidence of restenosis.

The essential aim of the invention is to inhibit restenosis by delivering a protein in a target blood vessel (e.g., artery or vein) that can inhibit the proliferation of VSMCs by the induction of cell cycle arrest (i.e., cellular senescence), and in some cases, the induction of apoptosis. The sustained release properties of nanoparticle formulations can maintain the low level of protein that maintains the cell-cycle arrest phase until the time that vascular repair occurs (i.e., re-endothelialization). Efforts are underway to develop cell/tissue specific, efficient, and safer gene expression vectors which can be used for arterial gene delivery to inhibit restenosis (1). However, the efficacy of gene therapy may be limited because it is not known how gene transfection occurs in the diseased cells. It is known that a significant number of VSMCs undergo apoptosis (in humans—20 to 30%, in rat carotid model—60 to 70%) in the injured artery within hours following angioplasty. In addition to the loss of cells that occurred due to angioplasty and apoptosis, a further loss of VSMCs due to cytotoxic drugs could lead to significant elastic recoiling of the artery. This results in a reduced lumen diameter, collapse of the artery or aneurysm causing arterial rupture, and bleeding. Moreover, greater loss of VSMCs provokes a greater body response to the injury (e.g. accumulation of platelets and secretion of growth factors) resulting in greater hyperplasia.

A better alternative to gene and drug therapy approaches could be a protein therapy, whereby a therapeutic protein is delivered to the target tissue or cells as shown, for example, in FIG. 1. With an effective delivery mechanism, one would be able to modulate more precisely the dose and the duration of protein delivery in the target artery to achieve inhibition of restenosis. Without being bound by theory, p53 plays central role in the control of cell growth and proliferation, and perhaps stabilizes VSMCs from undergoing apoptosis, thus, p53 can prevent the further cascade of events including inflammatory response that leads to hyperplasia. The fact that p53 levels are down regulated in the injured artery immediately following angioplasty and remain low during the proliferative phase of hyperplasia (10-14 days) provides a compelling evidence of its role in development of hyperplasia (2). Although p53 is known to affect the cell-cycle, as well as inducing apoptosis, its effect also depends on the level of gene expression and cell type. At a lower level of gene expression, p53 can inhibit cell proliferation primarily by cell-cycle arrest in G1 phase, whereas at higher levels, p53 can induce cell apoptosis (3,4).

p53 in Restenosis

The transcription factor, p53 regulates cell proliferation by multiple mechanisms

including increase in cell surface expression of the death ligand receptor Fas (5-8), or activation of apoptotic genes such as Bax (9), or cell-cycle arrest through the cyclin dependent kinase inhibitor p21 (10,11). The p53 protein acts as a checkpoint in the cell cycle, either preventing or initiating programmed cell death (apoptosis). p53 also switches on a series of protective genes when the cell is exposed to stressful events.

Inhibition of VSMC proliferation has been demonstrated with wt-p53 gene using hemagglutinating virus of Japan (HVJ) liposome complex (12), and adenoviral vector (13) in animal models. Recent studies have shown that p53 deficiency promotes atherogenesis in murine models in which atherosclerosis was induced by remnant-like lipoproteins with absence or dysfunctional apoE (11), which suggests a role of p53 in vascular proliferative response (14). p53 protein could also protect VSMCs from stress-induced apoptosis (from exposure to growth factors) following angioplasty because of its protective effect on cell genome (15). Therefore, with protein therapy, there could be reduced vascular recoiling, and the long-term patency of the artery will not be a concern. The efficacy of the approach would depend on achieving sustained protein transfection in the target artery that would inhibit the proliferation of VSMCs primarily by cell-cycle arrest. Thus, sustained release NPs could be more effective in our studies than other systems (e.g, lipid complexes) which show relatively higher but transient protein transfection.

p53 Protein vs. Gene Delivery

Using protein therapy, it should be possible to modulate the dose and duration of p53 effect in the target blood vessel (e.g., artery or vein) depending on the therapeutic response measured by inhibition of restenosis. This can be achieved by readjusting the NP formulation parameters for protein loading and its release profile. An exemplary formulation described herein contains only 0.4 pg protein per mg NPs (0.04% w/w loading). Notably, it is possible to load as much as 10,000 pg protein per mg NPs (10% w/w loading). Similarly, one can change the protein release rate and duration of release by selecting suitable polymer composition (lactide to glycoside ratio), and molecular weight in the formulation of NPs. Since p53 protein is more potent, the dose of NPs required in the target artery would be significantly lower than that would be required for a less potent drug such as dexamethasone. The lower dose of NPs would increase the efficiency of uptake of NPs by the target artery, and would reduce the down-stream flow of excess of NPs, hence, the protein therapy could be more “target specific”.

In previous studies using a porcine model, it was demonstrated that an increase in the arterial uptake of NPs with an increase in the dose of NPs infused was marginal beyond a certain dose, and the excess of the administered dose flows downstream (16). Potency of therapeutic agent is critical to developing an effective and target-specific NP-based system (or any other colloidal drug delivery system) for the inhibition of restenosis. Considering that the target artery has a limited holding capacity for NPs, it is necessary that the desired therapeutic dose of drug is delivered in the target artery in the dose of NPs that can be localized in the artery. This can be achieved using p53 protein because of its potency.

Furthermore, it is necessary that a therapeutic dose of protein is maintained in the target artery in order to prevent the proliferation of VSMCs for a period of time that would allow the injured artery to undergo the repair process.

As one of skill in the art will appreciate, a nanoparticle in accordance with the methods and compositions of the present invention can be composed of a variety of injectable biodegradable polymers. Nanoparticles are said to be biodegradable if the polymer of the nanoparticle dissolves or degrades within a period that is acceptable in the desired application (usually in vivo therapy), usually less than five years, and desirably less than one year, upon exposure to a physiological solution of pH 6-8 having a temperature of between 25° C. and 37° C. As such, a nanoparticle for use in accordance with the methods and compositions of the present invention can be composed of homopolymers or copolymers prepared from monomers of polymers disclosed herein, wherein the copolymer can be of diblock, triblock, or multiblock structure as described in U.S. Patent Application 20060067925. Suitable polymers include, but are not limited to, poly(lactide-co-glycolides), poly(lactic acid), poly(alkylene glycol), polybutylcyanoacrylate, poly(methylmethacrylate-co-methacrylic acid), poly-allylamine, polyanhydride, polyhydroxybutyric acid, or polyorthoesters and the like. In particular embodiments, a nanoparticle is composed of a copolymer of a poly(lactic acid) and a poly(lactide-co-glycolide). Particular combinations and ratios of polymers are well-known to the skilled artisan and any suitable combination can be used in the nanoparticle formulations of the present invention. Generally, the resulting nanoparticle typically ranges in size from between 1 nm and 1000 nm, or more desirably between 1 nm and 100 nm.

A nanoparticle of the present invention can further contain a polymer that affects the charge or lipophilicity or hydrophilicity of the particle. Any biocompatible hydrophilic polymer can be used for this purpose, including but not limited to, poly(vinyl alcohol).

To further enhance delivery of a therapeutically effective amount of an active agent, a nanoparticle of the present invention can further contain a targeting moiety (e.g., a protein transduction domain). As used herein, a targeting moiety is any molecule which can be operably attached to a nanoparticle of the present invention to facilitate, enhance, or increase the transport of the nanoparticle into target tissue. Such a moiety can be a protein, peptide or small molecule. For example, a variety of protein transduction domains, including the HIV-1 Tat transcription factor, Drosophila Antennapedia transcription factor, as well as the herpes simplex virus VP22 protein have been shown to facilitate transport of proteins into the cell (Wadia and Dowdy, (2002) Curr. Opin. Biotechnol. 13:52-56). Further, an arginine-rich peptide (Futaki, (2002) Int. J. Pharm. 245:1-7), a polylysine peptide containing Tat PTD (Hashida et al., (2004) Br. J. Cancer 90(6):1252-8); Deshayes et al., (2004) Biochemistry 43(6):1449-57) or an HSP70 protein or fragment thereof (WO 00/31113) is suitable for targeting a nanoparticle of the present invention. Not to be bound by theory, it is believed that such transport domains are highly basic and appear to interact strongly with the plasma membrane and subsequently enter cells via endocytosis (Wadia et al., (2004) Nat. Med. 10:310-315). Animal model studies indicate that chimeric proteins containing a protein transduction domain fused to a full-length protein or inhibitory peptide can protect against ischemic brain injury and neuronal apoptosis; attenuate hypertension; prevent acute inflammatory responses; and regulate long-term spatial memory responses (Blum and Dash, (2004) Learn. Mem. 11:239-243; May et al., (2000) Science 289:1550-1554; Rey et al., (2001) Circ. Res. 89:408-414; Denicourt and Dowdy, (2003) Trends Pharmacol. Sci. 24:216-218).

To conjugate or operably attach the targeting moiety to a nanoparticle of the present invention, standard methods such as the epoxy activation method can be employed. The nanoparticle surface is contacted with an epoxy compound (e.g., DENACOL®, Nagase America Co., CA) which reacts with the hydroxyl functional group of, e.g., the PVA associated with the nanoparticle surface. The epoxy activation of the nanoparticle creates multiple sites for reaction with a ligand and also serves as a linkage between the nanoparticle surface and the peptide to avoid steric hindrance for interaction of the peptide with the cell membrane (Labhasetwar et al., (1998) J. Pharm. Sci. 87:1229-34). The epoxy groups can react with many functional groups including amine, hydroxyl, carboxyl, aldehyde, and amide under suitable pH and buffer conditions; therefore increasing the number of possible targeting moieties which can be employed.

A nanoparticle formulation of the present invention can further contain a plasticizer to facilitate sustained release of the encapsulated active agent by maintaining the structure of the nanoparticle. Release of molecules (e.g., proteins, DNA or oligonucleotides) from nanoparticles formulated from block copolymers is, in general, not continuous. Typically, there is an initial release followed by a very slow and insignificant release thereafter. Not to be bound by theory, it is contemplated that the release profile may be as a result of the rapid initial drop in the molecular weight of the polymer which reduces the glass transition temperature of the polymer to below body temperature (37° C.); the glass transition temperature of copolymers prior to release is above body temperature (˜45 to 47° C.). Moreover, with degradation, these polymers become softer thereby closing the pores which are created during the initial release phase (due to the release of active agent from the surface). Therefore, an inert plasticizer is added to a nanoparticle formulation disclosed herein to maintain the glass transition temperature above 37° C. despite a decline in molecular weight of the polymer with time. In this manner, the pores remain open and facilitate a continuous release of the encapsulated active agent. Suitable plasticizers are generally inert and can be food/medical grade or non-toxic plasticizers including, but not limited to, triethyl citrate (e.g., CITROFLEX®, Morflex Inc., Greensboro, N.C.), glyceryl triacetate (e.g., Triacetin, Eastman Chemical Company, Kingsport, Tenn.), L-tartaric acid dimethyl ester (i.e., dimethyl tartrate, DMT) and the like. A particularly suitable plasticizer is L-tartaric acid dimethyl ester.

The amount of plasticizer employed in a nanoparticle composition can range from about 5% to 40% weight of the nanoparticle, more desirably from about 5% to 20% weight of the nanoparticle. In particular embodiments, the plasticizer encompasses about 10 weight percent of the nanoparticle composition.

By enhancing the release profile of an active agent, a plasticizer-containing nanoparticle has utility in the delivery of a variety of active agents to a variety of tissues or organs. Accordingly, the present invention further relates to a composition for sustained or continuous release of an effective amount of an active agent, for example p53 protein or shorter active fragments of p53 protein, wherein said composition contains an active agent, at least one biodegradable polymer, and an inert plasticizer. As used herein, “controlled release”, “sustained release”, and similar terms are used to denote a mode of active agent delivery that occurs when the active agent is released from the nanoparticle formulation at an ascertainable and controllable rate over a period of time, rather than dispersed immediately upon application or injection. Controlled or sustained release can extend for hours, days or months, and can vary as a function of numerous factors. For the composition of the present invention, the rate of release will depend on the type of the plasticizer selected and the concentration of the plasticizer in the composition. Another determinant of the rate of release is the rate of hydrolysis of the linkages between and within the polymers of the nanoparticle. Other factors determining the rate of release of an active agent from the present composition include particle size, acidity of the medium (either internal or external to the matrix) and physical and chemical properties of the active agent in the matrix.

As will be appreciated by the skilled artisan, the nanoparticle compositions of the present invention can further contain additional fillers, excipients, binders and the like depending on, for example, the route of administration and the active agent used. A generally recognized compendium of such ingredients and methods for using the same is Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro, editor, 20th ed. Lippingcott Williams & Wilkins: Philadelphia, Pa., 2000.

Definitions

The following definitions are provided to facilitate an understanding of the present invention:

The terms “p53”, “p53 protein”, or “p53 protein fragment” all refers to the nuclear protein that plays an essential role in the regulation of cell cycle, specifically in the transition from G0 to G1. p53 is a DNA-binding protein containing DNA-binding, oligomerization and transcription activation domains. It is postulated to bind as a tetramer to a p53-binding site and activate expression of downstream genes that inhibit growth and/or invasion, and thus function as a tumor suppressor. Mutants of p53 that frequently occur in a number of different human cancers fail to bind the consensus DNA binding site, and hence cause the loss of tumor suppressor activity. Exemplary “p53” proteins include the human p53, such as that listed by GenBank protein ID: NP_(—)000537, and its structural and functional polymorphisms. A list of p53 protein fragments for use in the NP formulations are listed in Table I. It has been suggested that p53 fragments lacking N- and/or C-terminal parts could have an effect on the regulation of p53 stability or function. The decoy p53 fragments can indirectly influence the function of p53. For example, it has been shown that mdm2 can promote the destabilization of p53 and that this function depends on interaction of both proteins. p53 decoy fragments can bind to mdm2 which can then make available the transcriptionally active p53. This could enhance the pro-apoptotic function of p53 in cancer treatment or its protective effect in normal cells from oxidative stress or radiation induced DNA damage (Kubbutal and Vousden, Molecular Medicine Today, June 1998, pgs. 250-256).

These non-functional p53 fragments discussed above may lack any known biological activity and can act as decoy molecules in the cell rather than inducing apoptosis or senescence. For example, the decoy p53 fragments could be delivered to suppress the activity of any mutated p53 protein, if present in the cell. Alternatively, the decoy fragments can be delivered to act as binding partners or substrates in the cell, thereby allowing wild type p53 to function normally in a particular cellular context.

As used herein, a “peptide”, “protein”, and “polypeptide” are used interchangeably and refer to a compound made up of a chain of amino acid residues linked by peptide bonds. The sequence for peptides is given in the order from the amino terminus to the carboxyl terminus. A peptide or peptide fragment is “derived from” a parent peptide or polypeptide if it has the amino acid sequence that is identical or homologous to the amino acid sequence of the parent peptide or polypeptide.

The term “isolated protein” or “isolated and purified protein” is sometimes used herein. This term refers primarily to a p53 protein of the invention, for example, those found in Table I. Alternatively, this term may refer to a protein that has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in “substantially pure” form. “Isolated” is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the fundamental activity, and that may be present, for example, due to incomplete purification, addition of stabilizers, or compounding into, for example, immunogenic preparations or pharmaceutically acceptable preparations.

The term “nanoparticle” refers to a particle having a size measured on the nanometer scale. As used herein, the “nanoparticle” refers to a particle having a matrix-type structure with a size of less than about 1,000 nanometers. When the nanoparticle includes a bioactive component, the bioactive component is entangled or embedded in the matrix-type structure of the nanoparticle. Nanoparticles include particles capable of containing a therapeutic/diagnostic agent that is to be released within a mammalian body, including specialized forms such as nanospheres, whether natural or artificial.

A “therapeutic agent” as used herein refers to an agent which can mitigate, cure, treat or prevent a disease or condition. It is particularly desirable that the therapeutic agent be capable of exerting it effect locally (i.e., at or near the site of the disease or condition). Exemplary therapeutic agents include, but are not limited to, antibiotics, anti-restenotics, anti-proliferative agents, anti-neoplastic agents, chemotherapeutic agents, cardiovascular agents, anti-inflammatory agents, immunosuppressive agents, anti-apoptotic and anti-tissue damage agents.

The term “delivery” as used herein refers to the introduction of foreign molecule (i.e., protein containing nanoparticle) in cells.

The phrase “blood vessel” refers to components of the circulatory system which functions to move blood throughout the body. This phrase includes both arteries, which move blood away from the heart, and veins, which circulate blood back to the heart.

The term “treating” as used herein means the prevention, reduction, partial or complete alleviation or cure of a disease.

The term “administration” as used herein means the introduction of a foreign molecule (i.e., protein containing nanoparticle) into a cell. The term is intended to be synonymous with the term “delivery”. Administration also refers to the methods of delivery of the compounds of the invention (e.g., routes of administration such as, without limitation, intravenous, intra-arterial, intramuscular, subcutaneous, intrasynovial, infusion, sublingual, transdermal, oral, or topical). The preferred method of delivery is to the blood vessel (e.g., artery or vein) or in particular applications to the carotid, coronary, femoral, renal, or cerebral artery, depending on the site of injury.

As used herein, an “effective amount” of the p53 protein or protein fragment is an amount sufficient to cause cell cycle arrest, or an amount sufficient to inhibit cell proliferation in a subject.

An “individual” as used herein refers to any vertebrate animal, preferably a mammal, and more preferably a human.

As used herein, “proliferating” and “proliferation” refer to cells undergoing mitosis. Throughout this application, the term “proliferative disorder” refers to any disease/disorder marked by unwanted or aberrant proliferation of tissue. As used herein, the term “cell proliferative disorder” refers to conditions in which the unregulated and/or abnormal growth of cells can lead to the development of an unwanted condition or disease, which can be cancerous or non-cancerous, for example a psoriatic condition.

The term “restenosis” refers to any pre-occlusive lesion that develops following a reconstructive interventional procedure such as balloon angioplasty or stenting in a diseased blood vessel. The term is not only applied to the recurrence of a pre-existing stenosis, but also to previously normal vessels such as vein grafts that become partially occluded following vascular bypass. Restenosis refers to any luminal narrowing that occurs following an injury to the vessel wall. Injuries resulting in restenosis can therefore include trauma to an atherosclerotic lesion (as seen with angioplasty), a resection of a lesion (as seen with endarterectomy), an external trauma (e.g., a cross-clamping injury), or a surgical anastomosis. Restenosis typically results from a hyperplasia. The loss of endothelium exposes the smooth muscle cells to growth factors, causing them to migrate and proliferate into the lumen of the artery. Restenosis is believed to occur in about 30% to 60% of lesions treated by angioplasty and about 20% of lesions treated with stents within 3 to 6 months following the procedure.

The term “inflammation” as used herein refers to the biologic response of body tissue to injury, irritation, or disease which can be caused by harmful stimuli, for example, pathogens, damaged cells, or irritants. Inflammation is typically characterized by pain and swelling. Inflammation is intended to encompass both acute responses, in which inflammatory processes are active (e.g., neutrophils and leukocytes), and chronic responses, which are marked by slow progress, a shift in the type of cell present at the site of inflammation, and the formation of connective tissue. The term “inflammation” also refers to “VSMC inflammation” in a patient following angioplasty.

Pharmaceutical Compositions

Methods of the invention directed to treating restenosis involve the administration of p53 protein containing nanoparticles. One skilled in the art appreciates that a p53 protein containing nanoparticle can be administered to a subject by various routes including, for example, orally or parenterally, such as intravenously (i.v.), intramuscularly, subcutaneously, intraorbitally, intranasally, intracapsularly, intraperitoneally (i.p.), intracisternally, intra-tracheally (i.t.), or intra-articularly or by passive or facilitated absorption, and most preferably, by injection.

Administration of the pharmaceutical preparation is preferably in an “effective amount” this being sufficient to show benefit to the individual. This amount prevents, alleviates, abates, or otherwise reduces the severity of symptoms in a patient.

The pharmaceutical preparation is formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to a physically discrete unit of the pharmaceutical preparation appropriate for the patient undergoing treatment. Each dosage should contain a quantity of active ingredient calculated to produce the desired effect in association with the selected pharmaceutical carrier. Procedures for determining the appropriate dosage unit are well known to those skilled in the art. Dosage units may be proportionately increased or decreased based on the patient and the desired effect. Appropriate concentrations for alleviation of a particular pathological condition may be determined by dosage concentration curve calculations, as known in the art.

Nanoparticle compositions provided herein can be prepared for local administration by a variety of different routes, including for example, directly to site of the disease or condition (e.g., a site of injury or tumor) under direct vision (e.g., at the time of surgery or via endoscopic procedures) or via percutaneous drug delivery to the exterior (adventitial) surface of the site of the disease or condition (e.g., perivascular delivery). As an alternative, the placement of nanoparticles via a catheter can also be accomplished.

Perivascular drug delivery involves percutaneous administration of the nanoparticle composition using a needle or catheter directed via ultrasound, computed tomography, fluoroscopic, positron emission tomography, magnetic resonance imaging or endoscopic guidance to the site of the disease or condition. Alternatively, the procedure can be performed intra-operatively under direct vision or with additional imaging guidance. In the case of restenosis or other cardiovascular diseases, such a procedure can also be performed in conjunction with endovascular procedures such as angioplasty, atherectomy, or stenting or in association with an operative arterial procedure such as endarterectomy, vessel or graft repair or graft insertion.

For example, in a patient with narrowing of the superficial femoral artery, balloon angioplasty would be performed in the usual manner (i.e., passing a balloon angioplasty catheter down the artery over a guide wire and inflating the balloon across the lesion). Prior to, at the time of, or after angioplasty, a needle would be inserted through the skin under ultrasound, fluoroscopic, or CT guidance and a therapeutic agent (e.g., p53 protein in a sustained-release nanoparticle) would be infiltrated through the needle or catheter in a circumferential manner directly around the area of narrowing in the artery. This could be performed around any artery, vein or graft, but ideal candidates for this intervention include diseases of the carotid, coronary, iliac, common femoral, superficial femoral and popliteal arteries and at the site of graft anastomosis. Logical venous sites include infiltration around veins in which indwelling catheters are inserted.

Nanoparticle compositions of the present invention can be administered either alone, or in combination with a pharmaceutically or physiologically acceptable carrier, excipient or diluent. Generally, such carriers should be nontoxic to recipients at the dosages and concentrations employed. Ordinarily, the preparation of such compositions entails combining the nanoparticle composition of the present invention with buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients.

Representative examples of restenosis therapeutic agents include, for example, anti-angiogenic agents such as anti-invasive factor (Eisentein et al., (1975) Am. J. Pathol. 81:337-346; Langer et al., (1976) Science 193:70-72; Horton et al., (1978) Science 199:1342-1345), retinoic acid and derivatives thereof which alter the metabolism of extracellular matrix components to inhibit angiogenesis, tissue inhibitor of metalloproteinase-1, tissue inhibitor of metalloproteinase-2, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, and anginex (Griffioen et al., (2001) Biochem. J. 354(Pt 2):233-42); collagen inhibitors such as halofuginone or batimistat; antisense oligonucleotides directed to nucleic acid sequences encoding c-myc or c-myb; growth factor inhibitors such as tranilast, trapidil or angiopeptin; antioxidants such as probucol, anti-thromobotics such as heparin or abciximab, anti-proliferative agents such as AG-1295 (Fishbein, et al. (2000) Arterioscler. Thromb. Vasc. Biol. 20:667), tyrphostin (Banai, et al. (2005) Biomaterials 26(4):451-61), pacitaxel or other taxanes (Scheller et al., (2004) Circulation 110(7):810-4), isoflavones (Kanellakis et al., (2004) Atherosclerosis 176(1):63-72), rapamycin or derivatives or analogs thereof (Schachner et al., (2004) Ann. Thorac. Surg. 77(5):1580-5), vincristine, vinblastine, HMG-CoA reductase inhibitors, doxorubicin, colchicines, actinomycin D, mitomycin C, cyclosporine, or mycophenolic acid; anti-inflammatory agents such as dexamethasone (Liu et al. (2004) Expert Rev. Cardiovasc. Ther. 2(5):653-60), methylprednisolone, or gamma interferon; and the like which exhibits antirestenotic activity.

Other therapeutic agents that can be utilized in accordance with the present invention include anti-proliferative, anti-neoplastic or chemotherapeutic agents to prevent or treat tumors. Representative examples of such agents include androgen inhibitors; antiestrogens and hormones (e.g., flutamide, leuprolide, tamoxifen, estradiol, estramustine, megestrol, diethylstilbestrol, testolactone, goserelin, medroxyprogesterone); cytotoxic agents (e.g., altretamine, bleomycin, busulfan, carboplatin, carmustine(BiCNU), cisplantin, cladribine, dacarbazine, dactinomycin, daunorubicin, doxorubicin, estramustine, etoposide, lomustine, cyclophosphamide, cytarabine, hydroxyurea, idarubicin, interferon alpha-2a and -2b, ifosfamide, mitoxantrone, mitomycin, paclitaxel, streptozocin, teniposide, thiotepa, vinblastine, vincristine, vinorelbine); antimetabolites and antimitotic agents (e.g., floxuridine, 5-fluorouracil, fluarabine, interferon alpha-2a and -2b, leucovorin, mercaptopurine, methotrexate, mitotane, plicamycin, thioguanine, colchicines); folate antagonists and other anti-metabolites; vinca alkaloids; nitrosoureas; DNA alkylating agents; purine antagonists and analogs; pyrimidine antagonists and analogs; alkyl solfonates; enzymes (e.g., asparaginase, pegaspargase); and toxins (e.g., ricin, abrin, diphtheria toxin, cholera toxin, gelonin, pokeweed antiviral protein, tritin, Shigella toxin, and Pseudomonas exotoxin A).

Further therapeutic agents that can be utilized within the present invention include cardiovascular agents such as antihypertensive agents; adrenergic blockers and stimulators (e.g., doxazosin, guanadrel, guanethidine, pheoxybenzamine, terazosin, clonidine, guanabenz); alpha-/beta-adrenergic blockers (e.g., labetalol); angiotensin converting enzyme (ACE) inhibitors (e.g., benazepril, catopril, lisinopril, ramipril); ACE-receptor antagonists (e.g., losartan); beta blockers (e.g., acebutolol, atenolol, carteolol, pindolol, propranolol, penbatolol, nadolol); calcium channel blockers (e.g., amiloride, bepridil, nifedipine, verapamil, nimodipine); antiaryythmics, groups I-IV (e.g., bretylium, lidocaine, mexiletine, quinidine, propranolol, verapamil, diltiazem, trichlormethiazide, metoprolol tartrate, carteolol hydrochloride); and miscellaneous antiarrythmics and cardiotonics (e.g., adenosine, digoxin, caffeine, dopamine hydrochloride, digitalis).

Additional therapeutic agents that can be used in accord with the present invention include anti-inflammatory agents. Representative examples of such agents include nonsteroidal agents (NSAIDS) such as salicylates, diclofenac, diflunisal, flurbiprofen, ibuprofen, indomethacin, mefenamic acid, nabumetone, naproxen, piroxicam, ketoprofen, ketorolac, sulindac, tolmetin. Other anti-inflammatory drugs include steroidal agents such as beclomethasone, betamethasone, cortisone, dexamethasone, fluocinolone, flunisolide, hydorcortisone, prednisolone, and prednisone. Immunosuppressive agents are also contemplated (e.g., adenocorticosteroids, cyclosporin).

Therapeutic agents also include anti-tissue damage agents. Representative examples of such agents include superoxide dismutase; immune modulators (e.g., lymphokines, monokines, interferon α and β); and growth regulators (e.g., IL-2, tumor necrosis factor, epithelial growth factor, somatrem, fibronectin, GM-CSF, CSF, platelet-derived growth factor, somatotropin, rG-CSF, epidermal growth factor, IGF-1).

As mentioned previously, a preferred embodiment of the invention comprises delivery of p53 protein containing nanoparticles to a patient in need thereof. P53 protein sequences and fragments of p53 for use in the invention are provided in Table I and the different domains of the p53 protein fragments are shown schematically in FIG. 2. The sequences in Table I include several p53 protein fragments (SEQ ID NOs: 1-18). Additionally, the p53 related protein p63 and p73, as well as the cell cycle proteins p21 and p27 can be delivered in nanoparticle formulations, and the sequences in Table II represent protein and protein fragments useful for the present invention.

TABLE I p53 (GenBank Accession number: NP_000537) protein sequences for use in nanoparticle formulations SEQ ID NO Description Reference Biological Activity 1 Full-length (1-393 aa) NP_000537 Induces apoptosis/senescence 2  1-186 aa JBC (2006) 281: 13566-13573 Destabilizes mitochondria membrane/ transcriptionally inactive (bax) 3 187-393 aa JBC (2006) 281: 13566-13573 Non-functional 4  1-110 aa JCI (2007) 117: 1008-1018 Non-functional 5  1-210 aa JCI (2007) 117: 1008-1018 Induces apoptosis via p73 6  1-312 aa JMB (2002) 322: 917-927 Binds DNA 7  1-323 aa MCB (1994) 14: 5182-5191 Suppresses ras transformation 8  1-343 aa PNAS (1994) 91: 1998-2002 No transcriptional repression/low growth suppression 9  1-355 aa Gene Dev. (1998) 12: 2831-2841 Reduced acetylation 10  22-186 aa JBC (2006) 281: 13566-13573 Destabilizes mitochondria membrane 11  22-393 aa JBC (2006) 281: 13566-13573 Destabilizes mitochondria membrane/ weak transcriptional activity (bax) 12  83-393 aa MCB (1994) 14: 5182-5191 No effect on ras transformation 13  93-393 aa JMB (2002) 322: 917-927 Binds DNA 14  94-312 aa JMB (2002) 322: 917-927 Binds DNA/ thermodynamically stable 15 100-393 aa PNAS (1994) 91: 1998-2002 Represses transcription/ low growth suppression 16 318-393 aa Gene Dev. (1998) 12: 2831-2841 Fully acetylated 17 340-393 aa MCB (1994) 14: 5182-5191 No effect on ras transformation 18 37-aa fragment JCI (2007) 117: 1008-1018 Induces apoptosis via Met + 118-142 + p73/binds to iASPP 171-181

TABLE II p21 (GenBank Accession number NP_000380), p27 (GenBank Accession number NP_004055), p63 (GenBank Accession number NP_003713), and p73 (GenBank Accession number NP_005418) protein sequences for use in nanoparticle formulations SEQ ID NO Description Reference 19 p21 (Full-length) 1-164 aa NP_000380 20 p21: (1-78 aa) US Appl. 2005/0032038 21 p21: (72-164 aa) US Appl. 2005/0032038 22 p27 (Full-length) 1-198 aa NP_004055 23 p27: (1-101 aa) US Appl. 2005/0032038 24 p27: (95-198 aa) US Appl. 2005/0032038 25 p73 (Full-length) 1-636 aa NP_005418 26 p73: 1-319 aa BBRC (2005), 333(3): 954-960 27 p73: 319-636 aa BBRC (2005), 333(3): 954-960 28 p63 (Full-length) 1-680 NP_003713

The materials and method set forth below are provided to facilitate the practice of the present invention.

Formulation and Characterization of Nanoparticles

PLGA (27 mg; 50:50, inherent viscosity 1.31; LACTEL, formerly Birmingham Polymers, Inc., Birmingham, Ala.) was dissolved in 1 mL of chloroform. Dimethyl tartrate (DMT or tartaric acid dimethyl ester; density 1.238 g/mL; Sigma, St. Louis, Mo.) 3 mg was dissolved in the polymer solution. Protein (10 mg of rat serum albumin (SIGMA A6272) and 20 μg of p53 protein (BD Pharmingen #556439)) was dissolved in 300 μL of water. The protein solution was emulsified into the PLGA solution by vortexing for 1 minute and then sonicating for 2 minutes at 55 Watts energy output using a probe sonicator (XL 2015 Sonicator® ultrasonic processor, Misonix, Inc., Farmingdale, N.Y.).

The resulting primary emulsion was further emulsified into 12 mL of 2% PVA solution (PVA average molecular weight 30,000-70,000) by vortexing followed by sonicating for 2 minutes at 55 Watts. PVA solution was filtered through a 0.22 micron syringe filter and saturated with chloroform prior to use. A few drops of chloroform were added at a time into the PVA solution, shaken and the supernatant was used for the formulation.

The emulsion was stirred overnight on a stir plate at room temperature followed by desiccation under vacuum for 1 hour. Nanoparticles thus formed were separated by centrifugation at 30,000 rpm for 30 minutes at 4° C. (Beckman OPTIMA® LE-80K, Beckman Instruments, Inc., Palo Alto, Calif.). Pelleted nanoparticles were resuspended in water and centrifuged again as indicated above. The supernatant was collected and the process was repeated one additional time to remove unencapsulated protein and emulsifier. The supernatants were collected and analyzed for protein levels to determine the amount of protein not encapsulated in the nanoparticles. Protein levels were determined using BIORAD® assay kit.

Nanoparticles were suspended in water by sonication as above. The suspension was lyophilized for 48 hours (VirTis Company, Inc. freeze dryer, Gardiner, N.Y.).

The diameter of the nanoparticles was obtained with photon correlation spectroscopy (PCS) using quasi elastic light scattering equipment (ZETAPLUS®, zeta potential analyzer, Brookhaven Instruments Corp., Holtsville, N.Y.) and ZETAPLUS® particle sizing software (version 2.07).

The following examples are provided to illustrate certain embodiments of the invention. In particular, the experiments that follow were performed to assess release of p53 from the nanoparticle formulation described herein. These examples are not intended to limit the invention in any way.

EXAMPLE I Sustained Release of p53 Protein From Nanoparticles

A western blot was performed to assess p53 release from the nanoparticle formulation (FIG. 3). The western blot analysis of p53 protein release from NPs demonstrated robust bands corresponding to the p53 protein band prior to its encapsulation. This confirms that the protein maintained its configuration following its encapsulating into the NPs, and also when it is released slowly from NPs.

EXAMPLE II Balloon Injury and Inhibition Restenosis With p53 Protein-Loaded NP in a Rat Carotid Artery Model

The preliminary study in rat carotid artery model demonstrated significant inhibition of restenosis with a single-dose localized administration of p53 protein-loaded NPs (dose of protein=1.6 microgram). After balloon injury, NP suspension in saline was infused over 5 minutes at 2 atm of pressure (three 1-min periods between infusions of 70 μl of the suspension, with a 1 min period between infusions). The control group contained NPs without p53 protein. After three weeks, infused arteries were isolated, sectioned every 3 mm from the proximal to the distal ends, and were analyzed for proliferation. See FIGS. 4A-4D. The data demonstrate that p53 protein in modified NPs is effective in inhibiting restenosis. There is significant inhibition of intima to media ratio (65% inhibition of restenosis), and a corresponding increase in the lumen diameter in the p53 protein treated animals as compared to that in control. The protein alone in solution was clearly not as effective that delivered in a nanoparticle formulation.

REFERENCES

-   1. P. Libby, Gene therapy of restenosis: promise and perils, Circ     Res 82 (1998) 404-6. -   2. D. W. Muller, The role of proto-oncogenes in coronary restenosis,     Prog Cardiovasc Dis 40 (1997) 117-28. -   3. K. Itahana, G. P. Dimri, E. Hara, Y. Itahana, Y. Zou, P. Y.     Desprez, and J. Campisi, A role for p53 in maintaining and     establishing the quiescence growth arrest in human cells, J Biol     Chem 277 (2002) 18206-14. -   4. L. Dubrez, J. L. Coil, A. Hurbin, F. de Fraipont, S. Lantejoul,     and M. C. Favrot, Cell cycle arrest is sufficient for p53-mediated     tumor regression, Gene Ther 8 (2001) 1705-12. -   5. M. Bennett, K. Macdonald, S. W. Chan, J. P. Luzio, R. Simari,     and P. Weissberg, Cell surface trafficking of Fas: a rapid mechanism     of p53-mediated apoptosis, Science 282 (1998) 290-3. -   6. H. Matsushita. R. Morishita, Aoki M. Tomita. Y. Taniyama. H.     Nakagami, T. Shimozato. J. Higaki. Y. Kaneda, and T. Ogihara,     Transfection of antisense p53 tumor suppressor gene     oligodeoxynucleotides into rat carotid artery results in abnormal     growth of vascular smooth muscle cells. Circulation 10 1(2000)     1447-52. -   7. D. Lin, M. T. Shields, S. J. Ullrich, E. Appella, and W. E.     Mercer, Growth arrest induced by wild-type p53 protein blocks cells     prior to or near the restriction point in late GI phase, Proc Natl     Acad Sci 89 (1992) 9210-4. -   8. K. Tanaka, J. P. Zou, K. Takeda, V. J. Ferrans, G. R.     Sandford, T. M. Johnson, T. Finkel, and S. E. Epstein, Effects of     human cytomegalovirus immediate-early proteins on p53-mediated     apoptosis in coronary artery smooth muscle cells, Circulation     99 (1999) 1656-9. -   9. C. Ihling, J. Haendeler, G. Menzel, R. D. Hess, G.     Fraednch, H. E. Schaefer, and A. M. Zeiher, Coexpression of p53 and     MDM2 in human atherosclerosis: implications for the regulation of     cellularity of atherosclerotic lesions. J Pathol 185 (1998) 303-12. -   10. W. S. el Deiry, T. Tokino, V. E. Velculescu, D. B. Levy, R.     Parsons, J. M. Trent, D. Lin, W. E. Mercer, K. W. Kinzler, and B.     Vogelstein, WAFI, a potential mediator of p53 tumor suppression,     Cell 75 (1993) 817-25. -   11. N. V. Guevara, H. S. Kim, E. I. Antonova, and L. Chan, The     absence of p53 accelerates atherosclerosis by increasing cell     proliferation in vivo, Nat Med 5 (1999) 335-9. -   12. Y. Yonemitsu, Y. Kaneda, S. Tanaka, Y. Nakashima, K. Komori, K.     Sugimachi, and K. Sueishi, Transfer of wild-type p53 gene     effectively inhibits vascular smooth muscle cell proliferation in     vitro and in vivo, Circ. Res 82 (1998) 147-56. -   13. M. Scheinman, E. Ascher, E. S. Levi, A. Hingorani, D. Shirazian,     and P. Seth, p53 gene transfer to the injured rat carotid artery     decreases neointimal formation, J Vasc Surg 29 (1999) 360-9. -   14. M. Aoki, R. Morishita, H. Matsushita, S. Hayashi, H.     Nakagami, K. Yamamoto, A. Moriguchi, Y. Kaneda, J. Higaki, and T.     Ogihara, Inhibition of the p53 tumor suppressor gene results in     growth of human aortic vascular smooth muscle cells. Potential role     of p53 in regulation of vascular smooth muscle cell growth,     Hypertension 34 (1999) 192-200. -   15. A. Aranda Anzaldo, F. Orozco Velasco, E. Garcia Villa, and P.     Gariglio, p53, is a rate-limiting factor in the repair of     higher-order DNA structure, Biochim Biophys Acta 1446 (1999) 181-92. -   16. V. Labhasetwar, C. Song, W. Humphrey, R. Shebuski, and R. J.     Levy, Arterial uptake of biodegradable nanoparticles: effect of     surface modifications, J Pharm Sci 87 (1998) 1229-34.

While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims. 

1. A method for inhibiting restenosis of a blood vessel comprising administering an effective amount of a protein containing nanoparticle via said blood vessel to a subject in need of treatment, thereby inhibiting restenosis in said blood vessel.
 2. The method of claim 1, wherein said protein is selected from the group consisting of p21, p27, p53, p63, p73, or a functional fragment thereof.
 3. The method of claim 2, wherein said protein is selected from the group of Table I or Table II.
 4. The method of claim 1, wherein the nanoparticle comprises a biodegradable polymer comprising a poly(lactide-co-glycolide), poly(lactic acid), poly(alkylene glycol), polybutylcyanoacrylate, poly(methylmethacrylate-co-methacrylic acid), poly-allylamine, polyanhydride, polyhydroxybutyric acid, or a polyorthoester or a combination thereof.
 5. The method of claim 1, wherein the nanoparticle further comprises a targeting moiety.
 6. The method of claim 1, wherein said blood vessel is an artery and is selected from the group consisting of carotid, coronary, femoral, renal, and cerebral.
 7. The method of claim 1, wherein the nanoparticle further comprises a plasticizer to facilitate sustained release of an antioxidant.
 8. The method of claim 7, wherein the plasticizer comprises L-tartaric acid dimethyl ester, triethyl citrate, or glyceryl triacetate.
 9. A p53 protein nanoparticle formulation for sustained release of an effective amount of p53 protein said formulation comprising p53 protein, at least one biodegradable polymer, and an inert plasticizer.
 10. The formulation of claim 9, further comprising at least one agent selected from the group consisting of an antioxidant, an anti-infective, an antiseptic, a steroid, a therapeutic peptide, an analgesic, an anti-inflammatory agent, an anticancer agent, a narcotic, an anesthetic, an antiangiogenic agent, a polysaccharide, a vaccine, an antigen, or a nucleic acid.
 11. The formulation of claim 9, wherein the biodegradable polymer comprises a poly(lactide-co-glycolide), poly(lactic acid), poly(alkylene glycol), polybutylcyanoacrylate, poly(methylmethacrylate-co-methacrylic acid), poly-allylamine, polyanhydride, polyhydroxybutyric acid, or a polyorthoester.
 12. The formulation of claim 9, wherein the plasticizer comprises L-tartaric acid dimethyl ester, triethyl citrate, or glyceryl triacetate.
 13. The formulation of claim 9, wherein the nanoparticle further comprises a targeting moiety.
 14. A method of managing VSMC inflammation in a patient following angioplasty comprising administering to said patient a therapeutic agent in an effective amount to manage VSMC inflammation.
 15. The method of claim 14, wherein said therapeutic agent is a protein containing nanoparticle formulation.
 16. The method of claim 15, wherein said protein containing nanoparticle formulation contains a protein or protein fragment set forth in Table I or Table II.
 17. A protein containing nanoparticle formulation wherein said protein is selected from the group consisting of SEQ ID NO: 1-28 in a pharmaceutically acceptable carrier.
 18. A method of inhibiting inflammation in a patient following angioplasty comprising administering to said patient a protein containing nanoparticle formulation comprising a protein or protein fragment selected from the group consisting of SEQ ID NO: 1-28, thereby inhibiting inflammation in said patient. 